Extended Data Fig. 7: Enhancing SPO11 dimerization stimulates DNA cleavage activity.

a, EMSA of binding to the ³²P-labeled oligonucleotide shown in Fig. 4f. A representative gel is shown at left, quantification at right (mean ± s.d. of n = 3 experiments). The apparent K_d (mean ± s.e.) can be viewed as an estimate of the affinity of binding for the first monomer complex. The cartoons are for illustration; we do not know the exact conformation(s) of doubly-bound complexes. b, No cleavage of the oligonucleotide (0.5 nM) by SPO11-Y138F complexes (10 nM). Performed once. c, Weak cleavage supported by Mg²⁺. Reactions contained 0.5 nM oligonucleotide, 10 nM wild-type SPO11 complexes, and 5 mM MgCl₂. Denaturing PAGE gel and quantification are provided (performed once). d, Nicking-only activity from mixture of Y138F and E224A mutant SPO11 complexes. Reactions contained 0.5 nM oligonucleotide, 5 nM of each mutant protein complex, and 5 mM MnCl₂. A representative gel is shown above, quantification is below (mean ± s.d. of n = 3 experiments). e, SPO11 covalently bound to nicked DNA. Reactions as in panel d were electrophoresed with or without prior digestion with proteinase K. Performed once. f, SEC profiles of FKBP-SPO11 or FRB-SPO11 complexes with TOP6BL. Coomassie-stained SDS-PAGE gels (above) and UV profiles (below) are shown for chromatography of anti-Flag affinity-purified material. Pooled fractions are indicated in red. This purification was conducted once. g, Mass photometry of purified FKBP-SPO11 and FRB-SPO11 complexes with TOP6BL. The blanks lacked protein; protein concentration in the lower graphs was 15 nM total (7.5 nM each fusion protein). Rapamycin was 5 µM when included. Particle counts (gray bars), gaussian fits (red lines), fitted mean ± s.d., and percentages of total particles are shown. Calculated masses are 123.0 kDa (FKBP) and 122.5 kDa (FRB) for monomeric and 245.5 kDa for dimeric complexes. Asterisks, background material also present in the blanks.

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